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Gating strategy and phosphorylation of P65 in response to CD40 stimulation. PBMCs of subjects were recovered from liquid nitrogen and stimulated with CD40L at concentrations ranging from 0 to1000 ng/ml, and phosphorylation of P65, P38, ERK, and JNK were monitored at different time points by instant fixation, permeabilization, and staining with Ab master mixes containing cell surface markers and phospho-epitope Abs. Single cells were then analyzed by flow cytometry. (A) Gating strategy to identify cell subtypes within PBMCs. Cells were first gated on forward light scatter area and side light scatter area to exclude cell debris. After singlet gating, cells were gated on CD3 versus CD4 to identify CD4 T cells (CD3+CD4+), CD8 T cells (CD3+CD4−), and monocytes (CD3dim CD4dim with higher side light scatter arrea than for lymphocytes). B cells were then identified in the CD3−CD4− population based on the presence of CD20 expression. Memory B cells (CD20+CD27+) and naive B cells (CD20+CD27−) were further identified in total B cell pools based on the presence or absence of CD27 expression. (B and C) Phosphorylation of P65 was analyzed in indicated cell subsets. The MFI was based on 2,000–40,000 single-cell measurements depending on cell type. (B) Overlaid histograms show the time course of P65 phosphorylation in memory and naive B cells of an HD after stimulation with 1 μg/ml CD40L. (C) Overlaid histograms show the dose response of P65 phosphorylation in memory and naive B cells at 15 min after stimulation with indicated concentrations of CD40L. Black histograms show the unstimulated samples corresponding to basal phosphorylation levels. Data are representative of nine healthy individuals tested.

Memory and naive B cells of RRMS_TN patients showed significantly higher phosphorylation of P65 (NF-κB) upon CD40 stimulation compared with HD. (A) PBMCs from RRMS_TN patients and HD were analyzed by multiparameter phosflow at different time points after stimulation with 2 ng/ml CD40L, gating on memory and naive B cell populations, as indicated. Scatter plots of the MFI for p-P65, p-P38, p-ERK, and p-JNK in memory and naive B cells are shown. Data shown are phosflow of PBMCs isolated from leukapheresis pack of RRMS_TN patients (n = 12, ●) and HD (n = 9, ○). (B) Western blot analysis of CD40-induced p-P65 and p-P38 in purified CD19+ B cells from HD and RRMS patients. CD19+ B cells were magnetically isolated from PBMCs from RRMS_TN patients (n = 6) and HD (n = 6) using CD19 microbeads. Isolated CD19+ B cells were left unstimulated (labeled as −) or stimulated with 2 ng/ml CD40L for 15 min (labeled as +). After stimulation, the lysates were harvested and analyzed by Western blot with Abs recognizing the phosphorylated form of P65 and P38 Abs. Level of β-actin was shown as a loading control. (C) Mean pixel values (band density) of Western blot data in (B) were acquired using Image Studio software. The levels of p-P65 and p-P38 were then normalized to β-actin level. Data shown are fold induction of p-P65 and p-P38 after CD40L stimulation compared with unstimulated condition.

Memory B cells of RRMS patients showed significantly higher phosphorylation of IKKα/β upon CD40 stimulation compared with HD. (A and B) PBMCs from RRMS patients (n = 12 with one data point from 12 RRMS patients, ●) and HD (n = 12 with two data points from 6 healthy individuals, ○) were analyzed by multiparameter phosflow at different time points after CD40L (2 ng/ml) stimulation, gating on memory (A) and naive (B) B cell populations, as indicated. Scatter plots of the MFI for p-IKKα/β are shown. Data shown are representative of two repeated experiments. Significant differences in MFI values were calculated by a Student t test (p < 0.05). (C–F) PBMCs from RRMS patients were stimulated with 2 ng/ml CD40L in the presence or absence of TCPA-1 inhibitor at indicated concentrations. The cells were instantly fixed 15 min after the stimulation and analyzed by phosflow to detect p-P65 and p-P38. Cells stimulated with CD40L in the presence of DMSO served as a vehicle control. Data shown are from one RRMS patient as a representative of experiments from three individual RRMS patients.

Phosphorylation level of P65 was reduced in B cells from RRMS patients after combination therapy with Avonex and Cellcept, and the p-P65 reduction correlates with changes in EDSS score in these patients. (A–D) Following CD40 stimulation for 15 min, the level of p-P65 and p-P38 in memory B (A and C) and naive B cells (B and D) from HD (n = 9, ○) and RRMS patients (n = 8) before (RRMS_TN [T0], ●) and after (RRMS_A+C [T1], ▴) the therapy were included. The levels of p-P65 and p-P38 were detected at 15 min after stimulation with CD40L at 2 ng/ml. The p values were calculated by a Student t test and are shown in the figure. (E and F) EDSS scores were calculated before (T0) and after (T1) the combination treatment with Avonex and Cellcept. Changes in EDSS score (ΔEDSS) were assessed by subtracting the EDSS at the time of sampling (EDSST1) with the baseline EDSS before any treatments (EDSST0). The peak MFI values for p-P65 (15 min after CD40 stimulation) in memory (A) and naive B cells (B) were plotted relative to ΔEDSS as indicated. RRMS_A+C, RRMS patients after therapy with Avonex and Cellcept; RRMS_TN, RRMS_TN patients before therapy.

Proposed model for dysregulation of CD40 signaling in B cells from RRMS patients. CD40–CD40L interaction recruits TRAFs (TRAF2, 3, 5, and 6), which bind to the CD40 cytoplasmic domain and mediate the activation of multiple signaling pathways, including canonical and noncanonical NF-κB pathways, the PI3K pathway, as well as phosphorylation of MAPKs, including P38, ERK, and JNK. For canonical NF-κB activation, TRAF2 or TRAF6 mediates signaling activation and phosphorylates the subunits (IKKα, IKKβ, and IKKγ) in the IKK complex, which further induces the phosphorylation of IκB. The phosphorylation of IκB induces its degradation, and this process leads to phosphorylation and nuclear translocation of NF-κB, where it acts as a transcription activator. In normal B cells (left), the CD40 signaling plays an essential role in B cell proliferation, survival, and cytokine production. In B cells of RRMS patients (right), we found enhanced CD40-mediated canonical NF-κB signaling whereas the MAPK pathway remains unaffected. A prediction of the model is that dysregulation of CD40-mediated NF-κB signaling leads to the hyperproliferation of MS B cells upon CD40 engagement as we described previously. Additionally, our data point to the potential of therapeutic interventions including Avonex/Cellcept combination therapy and GA therapy to correct this signaling dysregulation.